HBV X protein has been shown to have a transactivating effect on the expression of a variety of viral and cellular genes. The expression of tumor suppressor gene RB was studied in a transient expression system by transfecting HBV X gene into Hep G2 cells. The HBV X open reading frame was generated by PCR from plasmid containing wild type HBV DNA and was inserted into expression vector. The plasmid pHBV-X was used to transfect Hep G2, a human hepatoblastoma cell line. Protein was extracted 48 hours after DNA transfection. The level of endogenous RB protein was analyzed by western immunoblotting using RB monoclonal antibody. RB protein untransfected Hep G2 cells and Hep G2 transfected with vector alone were also analyzed as controls. The level of RB protein in pHBV-X transfected Hep G2 cells increased nearly 10-fold compared to the control cells. Preliminary in vitro co-immunoprecipitation studies between RB protein and HBV X protein do not indicate direct interaction between RB and HBV X proteins. The finding that HBV derived X gene can alter the expression of RB tumor suppressor gene suggest a possible role for the X gene in HBV mediated hepatocarcinogenesis. Work continues in an effort to determine the mechanism of transactivation by X protein. Host mediated transcriptional factor(s) involved in X-gene activities will be analyzed by X-protein transactivation effect in different hepatic and non hepatic cell lines.